Hindawi Publishing CorporationJournal of PharmaceuticsVolume 2013, Article ID 874875, 6 pageshttp://dx.doi.org/10.1155/2013/874875

Research ArticleAnalysis of Hydroxy Fatty Acids from the Pollen ofBrassicacampestris L. var. oleiferaDC. by UPLC-MS/MS

Nian-Yun Yang, Yi-Fang Yang, and Kun Li

Department of Traditional Chinese Medicine, Shanghai Institute of Pharmaceutical Industry, Shanghai 200040, China

Correspondence should be addressed to Yi-Fang Yang; yangyf4912@163.com

Received 31 May 2012; Revised 4 August 2012; Accepted 6 August 2012

Academic Editor: Anna Wesolowska

Copyright © 2013 Nian-Yun Yang et al.is is an open access article distributed under the Creative CommonsAttribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Ultraperformance liquid chromatography coupled with negative electrospray tandemmass spectrometry (UPLC-ESI-MS/MS) wasused to determine 7 hydroxy fatty acids in the pollen of Brassica campestris L. var. oleifera DC. All the investigated hydroxyfatty acids showed strong deprotonated molecular ions [M–H]−, which underwent two major fragment pathways of the allylscission and the 𝛽𝛽-�ssion of the alcoholic hydroxyl group. By comparison of their molecular ions and abundant fragment ionswith those of reference compounds, they were tentatively assigned as 15,16-dihydroxy-9Z,12Z-octadecadienoic acid (1), 10,11,12-trihydroxy-(7Z,14Z)-heptadecadienoic acid (2), 7,15,16-trihydroxy-9Z,12Z-octadecadienoic acid (3), 15,16-dihydroxy-9Z,12Z-octadecadienoic acid (4), 15-hydroxy-6Z,9Z,12Z-octadecatrienoic acid (5), 15-hydroxy-9Z,12Z- octadecadienoic acid (6), and 15-hydroxy-12Z-octadecaenoic acid (7), respectively. Compounds 3, 5, and 7 are reported for the �rst time.

1. Introduction

Pollen contains many kinds of nutrient which are necessaryto human body. It is well known as a natural nutrition,health food, and “perfect food.” e pollen preparation(Qian Lie Kang Tablets) made of Brassica campestris L. var.oleifera DC. has also been widely used in China as a goodtreatment for benign prostatic hyperplasia (BPH), and achemical study has found plenty of fatty acids in the pollen[1]. Reports have shown promising therapeutic effects offatty acids and their derivatives in treatment of BPH [2–6]. Hydroxy fatty acids contained in pollen are importantbioactive substances. Various kinds of hydroxy fatty acidsexhibit many pharmacological activities such as antitumor,antifungal, and prostaglandin E-like [7–9]. Although thepollen preparation has been used as phytotherapy for BPHfor a long time, its active components and mechanism ofaction remain unclear. e effects of the supercritical �uidextract and its residue of the plant were screened so asto clarify its active constituents, and its supercritical �uidextract decreased the size of the prostate of androgen-inducedprostatic rats by in vivo experiments (𝑃𝑃 𝑃 𝑃𝑃𝑃𝑃𝑃 and also

demonstrated remarkable inhibitory effects on 5𝛼𝛼-reductaseand aromatase through in vitro experiments. e chemicalinvestigation of its supercritical �uid extract has led to theisolation of some fatty acids and fatty acid derivatives [10],and the activity test experiments showed that fatty acidspossessed strong inhibitory activity on 5𝛼𝛼-reductase, whichwere consistent with literature reports [2–4]. e activitytest experiments also displayed that fatty acid derivativespossessed strong inhibitory activity on aromatase [10]. 5𝛼𝛼-reductase and aromatase are two important effect targetson prostate hyperplasia [11], so fatty acids and fatty acidderivatives play a coreaction role in treatment of BPH.UPLC-MS combine the efficient separation capability of UPLC andthe great power in structural characterization of MS andprovide new powerful approach to identify the constituentsin plant extracts rapidly and accurately. In this paper, weinvestigated the fragmentation behaviors of hydroxy fattyacids in aMicromass Q/TOFMass Spectrometer and empha-sized on the structural determination of hydroxy fatty acids1–7 (Figure 1) from the supercritical �uid extract by UPLC-ESI-MS/MS.

2 Journal of Pharmaceutics

OH

OOH

OH

OH

OOH

OH

OH

OH

OOHOH

OH

OH

OH

OH

O

OH

OH

O

OH

OH

O

OH

OH

O

1 2

3 4

5 6

7

F 1: Structural formulas of hydroxy fatty acids in the present study.

0

100

(%)

2 4 6 8 10 12 14 16 18 20

9.21

8.158.6

9.97

12.71

11.8

Time

11.1

1

34

5

6

7

22

F 2: Total ion chromatogram of supercritical �uid extract of pollen of Brassica campestris L. var. oleifera DC.

2. Materials andMethods

2.1. Standards and Reagents. Reference compounds, 15,16-dihydroxy-9Z,12Z-octadecadienoic acid (1) and 10,11,12-trihydroxy-(7Z,14Z)-heptadecadienoic acid (2), were iso-lated from the Pollen of B. campestris L. var. oleifera DC. bythe authors. eir structures were unambiguously identi�edby NMR techniques [10], and their purities were above 95%as determined by HPLC. HPLC-grade acetonitrile (MeCN)and methanol (AR grade) were obtained from Labscan (Stil-lorgan, Ireland), and the water used for UPLC was puri�edby a Milli-Q system (Millipore, Milford, MA, USA). Ethanolfor plant extraction was purchased from Shanghai ChemicalCorporation (Shanghai, China).

2.2. Plant Material and Sample Preparation. e pollen of B.campestris L. var. oleiferaDC. was collected from InnerMon-golia Autonomous Region of China in March 2004 and wasidenti�ed by Professor �u Feng of Jiangsu Botanic Institute. Avoucher specimen (BC-20060726)was kept in theHerbarium

of Shanghai Institute of Pharmaceutical Industry. e driedpowder (1 kg) of the rape pollen, of which the cell wall wasbroken by zymolysis, was extracted in supercritical �uid CO2with 95% ethanol as the cosolvent; a dark brown residue(52.3 g) was obtained. Two milligrams of the supercritical�uid extract were dissolved in 10 ml of methanol and �lteredover 0.45 𝜇𝜇mmicroporousmembrane forUPLC-MS analysis.

2.3. UPLC-MS Analysis. UPLC mass spectrometry was car-ried out on aWaters nanoACQUITYUPLC system combinedwith AMicromass Q/TOFMass Spectrometer via an electro-spray ionization interface. UPLC conditions are as follows:the column was an nanoACQUITY BEH130 C18 Columnand the column temperature was maintained at 30∘C; themobile phase was a gradient elution which was mixed withsolvents A (water) and B (acetonitrile).e gradient programwas as follows: initial 0–2min, using gradient elution A–B(98:2, v/v), 2–6min, linear change to A–B (65:35, v/v),6–12min, linear change to A–B (50:50, v/v), 12–15min,

Journal of Pharmaceutics 3

50

5789

100 150

183

200

223

235253

250 300 350

312

311

m/z

0

100(%

)

50

69

100 150

155

200

213

245

250

277278

295

300

313

314

350

0

100

(%)

m/z

50

89

100

115

150

183

200

213 239

250

255

269

279

281

300

327328

350

m/z

m/z

m/z

0

100

(%)

0

100

(%)

0

100

(%)

50

89

100 150 200

221223251

250

279291

300

309

310

350

m/z

m/z

0

100

(%)

0

100

(%)

50

73

100 150 200

207

221

250

251

275

293294

300 350 50

73

100 150

167

200

223

250

253

277

295

296

300 350

50

73

100

127

150 200

225

250

255

279

297

298

300 350

1 2

3 4

5 6

7

F 3: e product ion spectra of compounds 1–7.

4 Journal of Pharmaceutics

O

O

O

O

O

O

O

O

O

O

O

Om/z 309

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

O

−212 Da

−88 Da

−144 Da

m/z 115

m/z 239

m/z 183

O−

m/z 327

m/z 327

O−

O−

O−

O−

CH2

−

OHOH

OHOH

−58 Da

−

88 Da

−128 Da

m/z 311

m/z 311

m/z 253

m/z 223

m/z 183O−

O−

O−

O−

−68 Da

−158 Da

−100 Da

m/z 245

m/z 155

m/z 313O−

m/z 213m/z 313

O−

O−

−58 Da

−

88 Da

−86 Da

m/z 309

m/z 251

m/z 221

O−

m/z 223

O−

O−

O−

−42 Da

−72 Da

−86 Da

m/z 293

m/z 251

m/z 221

O−

m/z 207m/z 293

O−

O−

O−

−

42 Da

−

72 Da

−128 Da

m/z 295

m/z 253

m/z 223

O−

m/z 167

m/z 295

O−

O−

O−

−42 Da

−

72 Da

−170 Dam/z 297

m/z 255

m/z 225

O−

O−

O−

m/z 127

m/z 297

CH2

−

CH2

−

CH2

−

CH2

−

CH2

−

CH2−

OH

OH

OH

OHOH

OH

OH

OH OH

OH

OH

OH

OH

OH

OH

OH OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH

OH OH

OH

OH

OH

OH

OHOH

OHOH OHOH

O−

1

2

3

4

5

7

6

F 4: Characteristic fragmentation pathways for the molecular anion of compounds 1–7.

Journal of Pharmaceutics 5

linear change toA–B (40:60, v/v), 15–18min, linear change toA–B (20:80, v/v), and18–20min, linear change to A–B (0:100,v/v); the sample injection volume was 1𝜇𝜇L; the �ow rate was0.2mL⋅min−1. e Q/TOF MS condition was set as follows:ionizing source was electrospray ionization (negative ionmode); drying gas (N2) �ow rate was 10.0 L/min; drying gastemperature was 320∘C; capillary voltage was set to 3000V;fragmentation voltage was set to 120V; the full-scan secondorder mass spectra of the investigated compounds fromm/z50–350Da were measured using 500ms for collectiontime and three microscans were summed.

3. Results and Discussion

3.1. Mass Spectrometry Analysis of Reference Compounds 1-2. At �rst, the two reference compounds 15,16-dihydroxy-9Z,12Z-octadecadienoic acid (1), and 10,11,12-trihydroxy-(7Z, 14Z)-heptadecadienoic acid (2), were analyzed byUPLC-ESI-MS. Both hydroxy fatty acids showed strong[M–H]− ions, which is similar to the hydroxy fatty acidsreported in Tydemania expeditionis [12].

e full MS/MS product ion spectrum of compound1 was shown in Figure 3. Six primary product ions areobserved and it is proposed that they are formed in twomajor fragment pathways (Figure 4). Pathway I involves the𝛽𝛽-�ssion of alcoholic hydroxyl group along with the neutralloss of propionaldehyde and 2-hydroxy-butyraldehyde or 1-hydroxy-2-butanone, which results in the formation of m/z253 and m/z 223 as the major peaks. Pathway II involves theallyl scission leading to the formation ofm/z 183 as the mainpeak. Some other pathways indicated neutral loss of H2O orCO or H2 from the deprotonated molecular and fragments.e allyl scission of the molecular ion [M–H]− atm/z 313 of2 leads to the formation ofm/z 213, and the 𝛽𝛽 -�ssion of OHgroup of 2 lost 1,3-pentadiene and 2,3-dihydoxy-octa-5-enalor 1,3-dihydoxy-octa-5-en-2-one to produce themajor peaksofm/z 245 andm/z 155.

3.2. Structural Analysis of Hydroxy Fatty Acids in the Fin-gerprint Chromatogram. UPLC-ESI-MS was used to analyzethe chemical constituents of the supercritical �uid extractof pollen of B. campestris L. var. oleifera DC. (Figure 2),and the fragmentation behaviour of 7 hydroxy fatty acidswas analyzed by UPLC-ESI-MS/MS. In the total ion chro-matograms (Figure 2), the peaks at 9.21min and 9.97minshowed a molecular ion [M–H]− at m/z 311 and 313,respectively (Figure 3), which were identical to compounds1-2, respectively and con�rmed by the same characteristicdata of UPLC analysis. In the total ion chromatograms, peakat 8.15min showed a molecular ion [M–H]− at m/z 327, thepeak at 8.60min showed a molecular ion [M–H]− at m/z309, the peak at 11.10min showed a molecular ion [M–H]−at m/z 293, the peak at 11.80min showed a molecular ion[M–H]− at m/z 295, and the peak at 12.71min showed amolecular ion [M–H]− at m/z 297, which were identi�edby UHPLC-MS/MS analysis. From the product ion spectra(Figure 3) of compound 3 at 8.15min, compound 4 at8.60min, compound 5 at 11.10min, compound 6 at 11.80

min, and compound 7 at 12.71 min, it was found that theyshowed almost the same fragment pattern with compounds1-2.

e molecular ion of compound 3 indicated an excess of16Da in comparisonwith that of 1, which suggested a surplushydroxyl group in compound 2. e full MS/MS product ionspectrum (Figure 3) of compound 3 showed the characteristicproduct ions of m/z 239, 183, 269, 115, and 89, which wereformed from the allyl scission and the 𝛽𝛽-�ssion of OH grouprespectively (Figure 4). e fragment m/z 115 resulted froma 𝛽𝛽-�ssion of the OH group, which showed a hydroxyl groupon C-7. Some other pathways also displayed loss of H2O orCO from the deprotonated molecular and fragments. econ�guration of the ole�nic bonds was deduced from thebiosynthetic pathway, and the natural sources of unsaturatedfatty acids and their derivatives are rich in the cis isomer, sothe ole�nic bonds of compound 3 were Z geometry. us,compound 3 was tentatively assigned as 7,15,16-trihydroxy-9Z,12Z-octadecadienoic acid, and it is reported for the �rsttime.

e full MS/MS product ion spectrum (Figure 3) ofcompound 4 showed the characteristic product ions of m/z221, and 223, which were formed from the allyl scission, andanother major peak ofm/z 251 resulted from the 𝛽𝛽-�ssion ofthe OH group (Figure 4). ese characteristic product ionssuggested an additional ole�nic bond at C-6 in compound4 in comparison with 1. Some other pathways displayedloss of 18 Da or 30 Da from the deprotonated molecularand fragments. e con�guration of the ole�nic bondswas also determined from the biosynthetic pathway, andcompound 4 was tentatively assigned as 15,16-dihydroxy-9Z,12Z-octadecadienoic acid, and 15S,16S-4 was reported inthe literature [13].

e molecular ion of compound 5 indicated a lack of16Da in comparison with that of 4. e full MS/MS production spectrum (Figure 3) of compound 5 showed the charac-teristic peaks ofm/z 73, 207, 221 and 251.e fragment atm/z251 resulted from the𝛽𝛽-�ssion ofOHgroup (Figure 4), whichshowed a hydroxyl group on C-15. e con�guration of theole�nic bonds was also determined from the biosyntheticpathway, and compound 5 was tentatively assigned as 15-hydroxy-6Z,9Z,12Z-octadecatrienoic acid, and it is reportedfor the �rst time.

e molecular ion of compound 6 indicated a lackof 16Da in comparison with that of 1. e full MS/MSproduct ion spectrum (Figure 3) of compound 6 showedthe characteristic peaks of m/z 73, 167, 223, and 253. efragment at m/z 253 resulted from the -�ssion of OH group(Figure 4), which showed a hydroxyl group on C-15. econ�guration of the ole�nic bonds was also determined fromthe biosynthetic pathway, and compound 6 was tentativelyassigned as 15-hydroxy-9Z,12Z-octadecadienoic acid, and15R-6 was reported in the literature [14].

emolecular ion of compound 7 indicated an excess of 2Da in comparison with that of 6.e full MS/MS product ionspectrum (Figure 3) of compound 7 showed the characteristicpeaks of m/z 73, 127, 225, and 255. e fragment at m/z 255resulted from the 𝛽𝛽-�ssion of OH group (Figure 4), whichshowed a hydroxyl group on C-15. e con�guration of the

6 Journal of Pharmaceutics

ole�nic bonds was also determined from the biosyntheticpathway, and compound 7 was tentatively assigned as 15-hydroxy-12Z-octadecaenoic acid, and it is reported for the�rst time.

Acknowledgments

�is work was �nancially supported by the Natural Sci-ence Foundation (06ZR14078) and the Traditional Chi-nese Medicine Modernization Programme (08DZ1971801)of Shanghai Science and Technology Committee, which weregratefully acknowledged.

References

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[2] L. Y. Pérez, R. Menéndez, R. Má, and R. M. González, “Invitro effect of D-004, a lipid extract of the fruit of the cubanroyal palm (Roystonea regia), on prostate steroid 5𝛼𝛼-reductaseactivity,” Current erapeutic Research, vol. 67, no. 6, pp.396–405, 2006.

[3] M. L. Arruzazabala, R. Mas, D. Carbajal, and V. Molina, “D-004, a lipid extract from royal palm fruit, exhibits antidepressanteffects in the forced swim test and the tail suspension test inmice,” Drugs in R&D, vol. 6, pp. 281–289, 2005.

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[7] R. W. Jiang, M. E. Hay, C. R. Fairchild et al., “Antineoplasticunsaturated fatty acids from Fijian macroalgae,” Phytochem-istry, vol. 69, no. 13, pp. 2495–2500, 2008.

[8] H. Ohigashi, K. Kawazu, and H. Egawa, “Antifungal constituentof Sapium japonicum,” Agricultural and Biological Chemistry,vol. 36, pp. 1399–1403, 1972.

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[10] N. Y. Yang, K. Li, Y. F. Yang, and Y. H. Li, “Aromatase inhibitoryfatty acid derivatives from the pollen of Brassica campestris L.var. oleifera DC.,” Journal of Asian Natural Products Research,vol. 11, no. 2, pp. 132–137, 2009.

[11] L. L. Jin, K. Li, Y. F. Yang, and N. Y. Yang, “Advance inthe research of drug targets on prostate hyperplasia,” ChinesePharmaceutical Journal, vol. 49, pp. 161–165, 2009.

[12] J. L. Zhang, J. Kubanek, M. E. Hay, W. Aalbersberg, W. C. Ye,and R. W. Jiang, “Rapid identi�cation of triterpenoid sulfatesand hydroxy fatty acids including two new constituents fromTydemania expeditionis by liquid chromatography-mass spec-trometry,” Journal of Mass Spectrometry, vol. 46, pp. 908–916,2011.

[13] T. Caruso and A. Spinella, “First total synthesis of naturalaplyolides C and E, ichthyotoxic macrolides isolated fromthe skin of the marine mollusc Aplysia depilans,” TetrahedronAsymmetry, vol. 13, no. 19, pp. 2071–2073, 2002.

[14] M. Hamberg and G. Hamberg, “15(R)-hydroxylinoleic acid, anoxylipin from oat seeds,” Phytochemistry, vol. 42, no. 3, pp.729–732, 1996.

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